Influence of water and trehalose on α- and ß-relaxation of freeze-dried lysozyme formulations.

Molecular mobility in form of alpha and beta relaxations is considered crucial for characterization of amorphous lyophilizates and reflected in the transition temperatures Tgα and Tgß. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tgα and Tgß in amorphous freeze-dried samples. Lysozyme and trehalose as well as their mixtures in varying ratios were investigated. Three different residual moisture levels, ranging from roughly 0.5-7 % (w/w), were prepared via equilibration of the freeze-dried samples. Known plasticising effects of water on Tgα were confirmed, also via differential scanning calorimetry. In addition and contrary to expectations, an influence of water on the Tgß also was observed. On the other hand, an increasing amount of trehalose lowered Tgα but increased Tgß showing that Tgα and Tgß are not paired. The findings were interpreted with regard to their underlying molecular mechanisms and a correlation with the known influences of water and trehalose on stability. The results provide encouraging hints for future stability studies of freeze-dried protein formulations, which are urgently needed, not least for reasons of sustainability.

Characterization of caspase-2 inhibitors based on specific sites of caspase-2-mediated proteolysis.

Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1-6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors.

Structure-Based Design and Biological Evaluation of Novel Caspase-2 Inhibitors Based on the Peptide AcVDVAD-CHO and the Caspase-2-Mediated Tau Cleavage Sequence YKPVD314.

Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.